Ceruloplasmin (Cp) is the main Cu-containing protein in the blood plasma and some other body fluids. A GPI- linked form is tethered to the surface of some cells. The main interest in this protein has been its role as a mediator of cellular Fe release through interaction with ferroportin. However, there is strong evidence it has additional functions that include protection against radicals and delivering Cu to cells from the blood that may occur through receptors for Cp found on my cell surfaces. The objectives of the research here proposed are to determine definitively whether or not Cp delivers Cu directly to cells and by what mechanisms this occurs. We hypothesize that the mechanism would include binding to Cu transporter 1 (CTR1) and a Cu/Fe reductase (of the cytochrome b561 or Steap family), and that this results not only in release of Cu from Cp but the formation of apoCp. To test these ideas we will produce 64Cu-labeled mouse Cp in vivo, for use with mouse cell lines that do and do not express Ctr1; and produce labeled human Cp by cloning and expression in insect cells (or obtaining it from the secretions of human hepatoma cells) for use with human cell lines in which CTR1 has and has not been knocked down with siRNA. We will first determine that Cp-Cu is actually taken up (internalized) by the cultured cells, for which we already have strong preliminary evidence. Various approaches will be used to demonstrate internalization, including low pH washes and trypsinization of the cell surface, detection of 64Cu from Cp in the cytosol, monitoring of Cu entry by use of intracellular fluorescent copper probes, and rescue of SOD1 or cytochrome c oxidase activity (or down-regulation of CCS Cu chaperone protein levels) in deficient cells by incubation with holoCp. We will also test for formation of apoCp during the delivery process, and test the hypothesis that Cp interacts with both CTR1 and a reductase for uptake to occur. The reductase will be identified by screening cells for expression of likely candidates, and through chemical cross-linking, with verification by siRNA knockdown/overexpression, and demonstrating that excess non-radioactive Cu (II) and Fe (III) prevent uptake of 64Cu from Cp. Cross-linked proteins will be separated and identified by laser fragmentation and TOF-TOF mass spectrometry, and their identity functionally confirmed through overexpression and knockdown. Parallel studies in Ctr1 null and WT fibroblasts are expected to identify an unknown Cp-Cu uptake system in these cells. The results of our studies should provide definitive evidence on whether and by what means plasma Cp delvers Cu to mammalian cells, thus expanding our knowledge of the functions of this unique plasma protein beyond its role in Fe metabolism and transport. PUBLIC HEALTH RELEVANCE: Copper is a very important chemical element supporting the activities of numerous specific proteins, inside and outside of the cells of the human organism, from collagen to melanin, adrenaline and respiration, to the flow of iron out of cells. Ceruloplasmin is the most abundant copper-containing protein in the blood, and has multiple seemingly different functions. Most research on ceruloplasmin has focused on its role in iron homeostasis. This proposal focuses on testing the hypothesis that ceruloplasmin is an important transport protein for the delivery of copper directly to cells; it is also designed to determine exactly how transfer of copper from ceruloplasmin occurs, so we understand what may go wrong and how such problems might be circumvented to maintain healthy cells.